CN101492651B - Eukaryon recombinant plasmid containing prokaryon gene pprI and uses thereof - Google Patents

Abstract

The invention discloses a eukaryotic recombinant plasmid containing prokaryotic gene pprI; a recombinant carrier is pCMV-HA-pprI; the recombinant plasmid can enter the body of an animal by the electrotransfection of a living body to express a pprI gene protein and has remarkable radioresistance and curing effects on the acute severe radiation injury on animals; moreover, the recombinant plasmid has a half life and does not have toxic side effects.

Description

A kind of eukaryon recombinant plasmid that contains prokaryon gene pprI and uses thereof

Technical field

The invention belongs to the gene recombined vector field, relate in particular to eukaryon recombinant plasmid that contains prokaryon gene pprI and uses thereof.

Background technology

Along with the fast development of nuclear science technology, nuclear radiation has been widely used in every field such as national defence, industry, agricultural and medical science and has brought benefit to the mankind.Yet no matter in still wartime at ordinary times, heavy dose of ionizing rays that nuclear accidents such as nuclear explosion, nuclear terror, nuclear incident are produced can cause that to living organism serious acute radiation damages (ARI), and mortality ratio is high.The control of acute severe radiotherapy damage is one of the emphasis of international radiological medicine and national defence medical field research and difficult point always.

In recent years, develop rapidly and use along with what molecular biology and protein engineering were studied, cytokine has been tried out the clinical treatment in acute radiation injury.We studies show that, significantly reduction or low relatively of cytokine secretion is the one of the main reasons that radiotherapy damage is difficult to repair during radiotherapy damage.The lasting controllability of cytokine discharges, and may be the key of curing acute radiation injury.

The mode of application cell factor protein or plasmid is mainly subcutaneous or intramuscular injection at present, owing to cytokine albumen costs an arm and a leg, half life short increase frequency of injection or increases dosage in vivo, the easy serious toxic side effect of generation and patient can't be tolerated then.These drawbacks have limited the application of cytokine in radiotherapy damage is clinical.

Radioresistant bacterium (Deinococcus radiodurans) is aerobic-type, non-spore type, Gram-positive tetrad micrococci of being separated the rotten canned meat after radiation sterilization first by people such as U.S. scientist Arthur WAnderson in 1956, is one of the strongest microorganism of the radiation resistance found on the earth up to now.Because of it causes death and mutation effect has extremely strong resistance, the concern and the attention of extremely biomedical in recent years boundary and environmental engineering circle to ionizing rays, ultraviolet ray etc. are radiation-induced.Radioresistant bacterium can be repaired more than at least 150 dna double splitting of chain of every karyomit(e) (DSB) like clockwork in several hours behind irradiation.The radioresistant bacterium of exponential phase of growth is colibacillary 200 times to the resistance of ionizing rays, can tolerate hundreds of Gy ionizing radiation exposure.This extremely strong capability of resistance to radiation may be especially relevant to the extremely strong repair ability of double-stranded DNA fracture with the chromosomal ring-type imporosity of this bacterium, efficient accurate dna damage repair system, also may can protected protein (rather than DNA) to exempt from the oxidative damage that ionizing rays causes relevant with the low levels of the high-content of radioresistant bacterium kytoplasm manganese (cytosolicmanganese) and iron.

Short DNA repairs the protein induced factor of multiple-effect, and (inducer of pleiotropic proteins promotingDNA repair is a kind of at the newfound adjusting albumen of radioprotective coccus pprI).The pprI gene contains 987bp, 328 AA that encode, and the molecular weight of its product P prI is about 37KD.PprI plays crucial effect on anti-the putting of radioresistant bacterium, and the recA genetic transcription increases behind its energy stimulating radiation.PprI may quicken the reparation of DNA ionization radiation injury by the expression of regulating downstream genes such as recA, pprA as a switch gene.Studies show that the PprI albumen of radioresistant bacterium can make gamma-ray radiation resistance and the resistance of oxidation all obviously raising of intestinal bacteria to high dosage at expression in escherichia coli.

Radioprotective Pseudomonas prokaryotic organism exist greatest differences with the eukaryote Mammals on evolving, genomic constitution is different with protein expression, and has the preferences problem of codon.Therefore, to express in mammalian cell almost be impossible to prokaryotic gene.Mammalian cell does not contain pprI gene or homology analogue, can can radioresistant bacterium pprI gene be imported mammalian cell so, and this gene express and bring into play the effect of its Antiradiation injury? this science difficult problem is not seen relevant report in the document at home and abroad so far as yet.

Summary of the invention

The object of the invention provides a kind of eukaryon recombinant plasmid that contains prokaryon gene pprI, thereby makes pprI prokaryotic gene successful expression in eukaryotic cell, realizes leap and the protein expression of pprI gene between two macrospecies system; Another object of the present invention provides the purposes of the eukaryon recombinant plasmid that contains prokaryon gene pprI.

For achieving the above object, the concrete technical scheme of the present invention is that a kind of eukaryon recombinant plasmid that contains prokaryon gene pprI, described recombinant plasmid are pCMV-HA-pprI, this plasmid is transformed into preservation in the intestinal bacteria, and its preservation information is: depositary institution: Chinese typical culture collection center; Address: Chinese Wuhan University; Preservation date on December 11st, 2008; Deposit number CCTCC M 208253; Classification name: bacillus coli DH 5 alpha/pCMV-HA-pprI; Escherichia coli DH5 α/pCMV-HA-pprI;

PCMV-HA-pprI construction of recombinant plasmid in the technique scheme may further comprise the steps:

(1) be upstream primer with 5 '-ATGCCCAGTGCCAACGTCAGCCCCCCTT-3 ', 5 '-TCACTGTGCAGCGTCCTGCGGCTCGTCC-3 ' is a downstream primer, is template with D.radiodurans R1 genome, and pcr amplification obtains the pprI gene;

(2) recycling step (1) gained PCR product is connected with the pGEM-T carrier, will connect product transformed into escherichia coli DH5 α, selects positive colony then, the extracting plasmid;

(3) with 5 '-TC GAATTCCCAGTGCCAACGTCAGCCCCCCTTGC-3 ' is a upstream primer, and the part of wherein ruling GAATTC is the EcoRI restriction enzyme site; With 5 '-TT CTCGAGTTTCACTGTGCAGCGTCCTGCGGCTC-3 ' is a downstream primer, and the portion C of wherein ruling TCGAG is the xhoI restriction enzyme site; With the pGEM-T-pprI plasmid that checks order correct is template, uses pcr amplification, and the rubber tapping purifying reclaims the purpose fragment;

(4) with EcoRI enzyme and XhoI enzyme pprI fragment and pCMV-HA carrier are carried out double digestion, after purifying enzyme is cut product, with the T4 ligase enzyme pprI fragment and pCMV-HA carrier were carried out ligation in 5: 1 in molar ratio, to connect product transformed into escherichia coli DH5 α, and coat on the LB flat board of penbritin and cultivate, the picking positive colony is done and is boiled bacterium PCR checking, and the extracting plasmid carries out dna sequencing, and correct clone protects bacterium with order-checking.

In the technique scheme, described pCMV-HA carrier is existing carrier, and its structural representation is seen Fig. 4; The sequence of the multiple clone site of carrier (MCS) and wherein the restriction enzyme site position is as follows:

The present invention also comprises the described application of eukaryon recombinant plasmid in the preparation antiradiation injury medicine that contains prokaryon gene pprI.

The present invention also comprises the medicine of a kind of prevention or treatment radiation injury, and described medicine contains the described eukaryon recombinant plasmid that contains prokaryon gene pprI of claim 1.

Recombinant plasmid pCMV-HA-pprI is stored in the bacillus coli DH 5 alpha among the present invention, extracting plasmid method: connect bacillus coli DH 5 alpha and contain in the LB liquid nutrient medium of penbritin of 100ug/ml in 5ml, after shaking bacterium 12h, get final product with the extracting of plasmid a small amount of extraction agent box.

Because the technique scheme utilization, the present invention has following advantage:

(1) the present invention uses two pairs of upstream and downstream primers of design voluntarily, successfully made up eukaryon recombinant plasmid PCMV-HA-pprI, this carrier can have been captured prokaryotic gene is difficult to successfully transfection and expression at eukaryotic cell a science difficult problem successfully at mammalian cell expression Pprl albumen.

(2) the eukaryon recombinant plasmid PCMV-HA-pprI of the present invention's structure the animal acute radiation injury is had significant prevention and treatment effect, and half life, is long, has no side effect behind the live body electrotransfection.

Description of drawings

Depositary institution: Chinese typical culture collection center; Address: Chinese Wuhan University; Preservation date on December 11st, 2008; Deposit number CCTCC M 208253; Classification name: bacillus coli DH 5 alpha/pCMV-HA-pprI; Escherichia coli DH5 α/pCMV-HA-pprI;

Fig. 1. be that the template pcr amplification obtains pprI fragment electrophorogram with D.radiodurans R1 genome among the embodiment one;

Wherein, M:DL2000Marker; The about 1000bp of 1:pprI fragment;

Fig. 2. boil bacterium PCR checking result among the embodiment one;

Wherein, 3: positive colony, M:DL2000Marker;

Fig. 3. detect the expression electrophorogram of pprI albumen in the 293T cell with Western blotting among the embodiment two;

Wherein, pCMV-HA-pprI is a recombinant plasmid, and pCMV-HA is an empty carrier, and the PprI albumen of expression is about 37KD;

Fig. 4. the structural representation of pCMV-HA carrier in the embodiment of the invention.

Embodiment

Below in conjunction with drawings and Examples the present invention is further described:

Experiment material:

PGEM-T carrier (Promega), pCMV-HA carrier (Clontech), Taq archaeal dna polymerase (flute river, Shanghai bio tech ltd), PCR product purification test kit (go up marine Ke Yingda biotech company), restriction enzyme EcoRI, xhoI (BioLabs company), dna ligation kit (Promega company), sepharose reclaims purification kit (QIAGEN) plasmid extraction test kit (Roche) in a small amount, Lipofectinamine2000 (invitrogen), the high sugar of calf serum and DMEM (Gibco), the mouse monoclonal antibody of HA-Tag (Cell Signaling Technology), mountain sheep anti-mouse igg (H+L) HRP binding substances (Cell Signaling Technology).

Radioresistant bacterium D.radiodurans R1 is incubated in TGY (0.5% Tryptones, 0.1% glucose, 0.3% yeast extract, wherein percentage ratio the is massfraction) substratum at 30 ℃; Preserve this chamber of bacillus coli DH 5 alpha, and 37 ℃ are incubated in LB (1%NaCl, wherein percentage ratio is massfraction for 1%Typtone, the 0.5%Yeast extract) substratum; HEKC 293T is at 37 ℃, 5%CO ₂Cultivate with the high sugar of the DMEM that contains 10% calf serum in the incubator.

Embodiment one, the pCMV-HA-pprI construction of recombinant plasmid

(1) clone of pprI gene

Extract the D.radioduransR1 genomic dna, clone primer according to this genome sequence design:

Upstream primer: 5 '-ATGCCCAGTGCCAACGTCAGCCCCCCTT-3 '

Downstream primer: 5 '-TCACTGTGCAGCGTCCTGCGGCTCGTCC-3 '

With D.radiodurans R1 genome is that template is carried out pcr amplification, amplification condition: 94 ℃ of 5min; 94 ℃ of 1min, 54 ℃ of 1min, 72 ℃ of 1min, 35 circulations; 72 ℃ of 10min.

Expand the purpose band and be about 1kb, reclaim the back through rubber tapping and detect, obtain the band (Fig. 1) of 1 clauses and subclauses with 1% agarose gel electrophoresis, and with PCR product purification test kit purified product with quantitative.

(2) structure of PCMV-HA-pprI carrier

2.1 the structure of pGEM-T-pprI plasmid:

With the PCR product after rubber tapping is reclaimed, purpose band and pGEM-T carrier are carried out ligation, 16 ℃ are spent the night, to connect product transformed into escherichia coli DH5 α and coat the LB of the penbritin that contains 100ug/ml and cultivate behind the 12h picking positive colony and do and boil bacterium PCR checking for dull and stereotyped last 37 ℃, and will verify that the positive clone's extracting plasmid of result carries out dna sequencing.

2.2pCMV-HA-pprI construction of recombinant plasmid:

With the pGEM-T-pprI plasmid that checks order correct is template, designs the following primer that contains restriction enzyme site:

Upstream primer is: 5 '-TC GAATTCCCAGTGCCAACGTCAGCCCCCCTTGC-3 ', the line part is the EcoRI restriction enzyme site; Downstream primer is 5 '-TT CTCGAGTTTCACTGTGCAGCGTCCTGCGGCTC-3 ', the line part is the xhoI restriction enzyme site.

Use pcr amplification, and the rubber tapping purifying reclaims the purpose fragment.With EcoRI enzyme and XhoI enzyme pprI fragment and pCMV-HA carrier are carried out double digestion, after purifying enzyme is cut product, 1% agarose gel electrophoresis is quantitative, pprI fragment and carrier are carried out ligation by 5: 1 (mol ratio), to connect product transformed into escherichia coli DH5 α and coat on the LB flat board of penbritin and cultivate, the picking positive colony is done and is boiled bacterium PCR checking, as seen No. 3 positive clones, it expands and single purpose band (Fig. 2), after getting No. 3 clone inoculation culture 12h, take out plasmid and order-checking, the forward sequencing result is shown in SEQ ID NO.1 in the sequence table, and the backward sequencing result is shown in SEQ ID NO.2 in the sequence table; Sequencing result is compared with NCBI blast-n, and the result shows consistent with the nucleotide sequence of pprI (NCBI: be numbered DR 0167), and correct clone protects bacterium with order-checking.

Embodiment two, and pCMV-HA-pprI plasmid transfection 293T cell and expression are identified

With the 293T cell with 1 * 10 ⁵The density of cells/ml, the amount of 1ml is seeded in the Tissue Culture Dish of 35mm, DMEM culture medium culturing 18h with 10% calf serum, after treating cell growth remittance sheet 70%-80%, cell culture medium is changed to serum free medium optimen (Gibco), the PCMV-HA-pprI plasmid of 1 μ g and the Lipofectinamine2000 liposome of 3 μ l are changed in the 293T cell, the empty carrier that changes 1ug simultaneously in contrast again.Behind the 4-6h serum free medium optimen is changed to the DMEM that contains 10% calf serum, cultivates 24h.

Western blotting detects the proteic expression of pprI: with cell centrifugation, use the cell pyrolysis liquid cracking, do the SDS-PAGE electrophoresis with getting 20ul behind its supernatant and the sample-loading buffer mixing, albumen is gone to cellulose nitrate film from polyacrylamide gel, film is placed the sealing damping fluid of 20ml with the configuration of 5% skim-milk, place shaking table concussion 1.5h under the room temperature; The antibody of mouse-anti HA was diluted in 5% skim-milk with 1: 1000, and 4 ℃ of shaking table concussions are spent the night.To resist mouse two to resist by 1: 5000 and be diluted in 5% skim-milk, place the shaking table concussion to hatch 1h under the room temperature, colour developing, X sheet exposure imaging, the result shows: can detect the proteic expression of PprI in the cells transfected, be about 37KD, as shown in Figure 3; Transfection the cell of empty carrier then do not detect the proteic expression of PprI.

This shows that the PprI albumen of prokaryotic organism radioresistant bacterium can be expressed in eukaryotic cell 293T.

Embodiment three, and pCMV-HA-pprI recombinant plasmid live body electrotransfection is to the preventive and therapeutic effect of acute severe radiotherapy damage

1.1 laboratory animal and grouping

Adopt the male BALB/c mouse of cleaning level pure strain, body weight 18 ± 2g is provided by Sichuan University's medical board Experimental Animal Center.Raise through all left and right sides adaptive phase, BALB/c mouse is divided into normal control group, simple irradiation group, transgenosis medicine group at random.

Simple irradiation group and the winding of transgenosis medicine are subjected to neutron or gammairradiation, place sterilisable chamber to raise according to the back mouse; And, put to death 4 mouse samplings for every group and detect in shining the back the 1st, 7,14,21 and 28 day.

1.2pprI dna murine live body electrotransfection

Transgenosis group mouse is in pre-irradiation 24h, the pCMV-HA-pprI plasmid 50 μ g/50 μ l TE liquid that the flesh intramuscular injection makes up voluntarily before the mouse thigh, the live body gene electrical transduction technology of optimizing application gives 8 of the electricimpulses of strength of electric field 200v/cm, time length 20ms and frequency 1Hz.

1.3 the making of chmice acute severe radiotherapy damage model

1.3.1 neutron radiation

Adopt Mianyang, Sichuan Chinese Academy of Sciences's physics and chemistry institute K-400 type DT neutron producer, neutron energy is 14MeV, mouse full-body exposure, absorption dose are respectively 0.2Gy, 0.6Gy, 1.0Gy and 2.0Gy, and wherein the 0.6Gy component is simple irradiation group and transgenosis medicine group.

1.3.2 gammairradiation

Adopt ⁶⁰The Co gamma-rays, the mouse full-body exposure, dose rate is 1Gy/min, absorption dose is 6Gy.

1.4pprI dna murine live body electrotransfection prevention effect is observed

1.4.1 mouse death rate is observed: mortality ratio observation group mouse is divided into 4 groups at random and carries out the whole body neutron irradiation, absorption dose and size of animal be respectively 0.2Gy8 only, 0.6Gy16 only, 1.0Gy8 only and 2.0Gy20 only.Irradiation back mouse places sterilisable chamber to feed, and observes the time and the quantity that were subjected in 30 days according to dead mouse, calculates mouse death rate.Table 1 is listed in the variation of various dose neutron radiation induced mice mortality ratio.

The variation of table 1 neutron radiation mouse death rate

By table 1 as seen, 30 days mortality ratio of mouse increase with the increase of neutron radiation whole body absorption dose.0.6Gy neutron causes chmice acute severe radiotherapy damage, transgenosis group mouse death rate (12.5%) is starkly lower than simple irradiation group (37.5%).2.0Gy irradiation group mouse is in shining back all death in the 4th, 5 day.

1.4.2 histopathology

Get simple irradiation group, transgenosis group mouse liver, kidney, testis and fix, conventional dehydration, embedding, 5 μ m section, HE dyeing, om observation with 10% neutral formalin.

The liver organization pathology change: shone the back the 1st day, congested, expansion appears in blood sinus around the simple irradiation group mouse liver leaflet central vein, wall of vein is not seen considerable change, liver cell sex change occurs and a small amount of necrosis is arranged, central vein was significantly congested in the 7th day, and wall of vein begins to have the collegen filament calmness, and large stretch of sex change, necrosis appear in liver cell, in more extensive according to the 14th day hepatic necrosis in back, and proliferation of fibrous tissue appears.The transgenosis group is in the liver cell oedema occurring on the 1st day according to the back, and compare degree of necrosis with control group obviously slight, and seldom downright bad, oedema increased the weight of in the 7th day, and less necrosis is arranged, and beginning in the 14th day obviously recovers, and basic recovery was normal in the 28th day.

The renal tissue pathology change: shone the back the 1st day, simple irradiation group mouse kidney does not see that obvious pathology change, and the renal glomerulus form is regular, and the uriniferous tubules no abnormality seen is not also seen hyperemia or oedema in the medullary substance.The 7th day, slight vitreous degeneration appearred in glomerular capillary, also saw small amount of liquid in the little balloon cavity, also occurred vitreous degeneration in the collecting tubule, and medullary substance has oedema.According to back the 14th day, the pathological change of kidney each several part obviously increased the weight of than the 7th day, and glomerular capillary tangible vitreous degeneration occurs and accompanies blood plasma to invade profit, and the medullary substance oedema increases the weight of.Transgenosis group mouse did not also see that obvious pathological change was arranged on the 1st day according to the back, and vitreous degeneration appearred in glomerular capillary in the 7th day, but simple irradiation group is light, and medullary substance not water breakthrough swells, and the 14th day begins to occur recovering, the basic recovery normally to the 28th day.

1.4.3 the peripheral blood leucocyte counting is observed

Eyeball of mouse is got blood, places the heparin tube that contains EDTA, gets in the acetate that 20ul adds 0.38ml 2%, mixing, irritates plate and leaves standstill 5min, carries out white blood cell count(WBC) under the low power lens.

Table 2 is listed in the variation of each experimental mice peripheral blood leucocyte sum.

The variation of different time peripheral blood leucocyte after table 2. mouse is shone (x ± s)

Annotate: compare with control group ^*P＜0.05, ^*P＜0.01; Compare #P＜0.05, ##P＜0.01 with simple control group.

By table 2 as seen, occur obviously reducing, reduced to Schwellenwert on the 7th day, began the appearance recovery on the 14th day according to the back according to the back according to back first day simple irradiation group and transgenosis group white blood cell count(WBC).Each time point transgenosis group white blood cell count(WBC) all is higher than simple irradiation group, wherein has highly significant significant difference (P＜0.01) on the the 1st, 14,21 and 28 day.

1.4.4 peripheral blood leucocyte counting and lymphocyte classification are observed

Eyeball of mouse is got blood, and the preparation blood smear is used Wright's stain dyeing, carries out the lymphocyte differential count under the mirror.Table 3 is listed in the percentile variation of different time peripheral blood lymphocyte after the neutron irradiation.

The percentile variation of different time peripheral blood lymphocyte after table 3. mouse is shone (x ± s)

Annotate: compare * P＜0.05 with control group, * * P＜0.01; Compare #P＜0.05, ##P＜0.01 with simple control group

By table 3 as seen, compare with control group, simple irradiation group and transgenosis group lymphocyte percentage all significantly reduce in the observation period, have significance or highly significant significant difference (P＜0.05 or 0.01).Simple irradiation group and transgenosis group lymphocyte percentage were reduced to Schwellenwert on the 7th day in reducing in the 1st day according to the back, and beginning in 14 days slowly recovers.Compare with simple irradiation group, transgenosis group lymphocyte percentage all is higher than simple irradiation group, but does not see significant difference (P＞0.05).

1.4.5 medullary cell, spleen and thymic lymphocytes apoptosis detect

Mouse is put to death and gets spleen and thymus gland, scrapes legal system with comb and is equipped with the lymphocyte single cell suspension.Get femur, wash medullary space with PBS, preparation medullary cell single cell suspension with the 1ml syringe.Cell suspension is centrifugal, and (1500rpm, 10min), supernatant discarded is with cold PBS suspension cell again.The recentrifuge cell, supernatant discarded is used 1x Annexin-V binding buffer liquid re-suspended cell, adjusts cell concn to 1 * 10 ⁶Individual/ml.The cell suspension of every 100ul adds Alexa Fluor488 Annexin-V and the 1ul PI working fluid (100ug/ml) of 5ul, incubated at room 15min.Add the 1x Annexin-V binding buffer liquid of 400ul in the cell suspension, put on ice mixing sample gently.Use flow cytometer (U.S. company BD) and carry out the apoptosis detection, the result represents with apoptosis rate (%).

Each experimental mice neutron is listed in table 4 according to the variation of back apoptosis in bone marrow cells.

The variation of different time apoptosis in bone marrow cells after table 4 mouse is shone (x ± s)

Annotate: compare with control group ^*P＜0.05, ^*P＜0.01; Compare #P＜0.05, ##P＜0.01 with simple control group

By table 4 as seen, with control group relatively, neutron was according to back the 1st day, simple irradiation group apoptosis in bone marrow cells occurs significantly increasing (p＜0.01), raises but no difference of science of statistics and transgenosis group apoptosis rate is slight; According to back the 7th day, simple irradiation group and transgenosis group apoptosis in bone marrow cells all significantly increased and reach maximum value; According to back 14d, simple irradiation group and transgenosis group medullary cell apoptosis lower gradually; 28d, transgenosis group apoptosis in bone marrow cells significantly reduces, with the control group no difference of science of statistics.Transgenosis group apoptosis in bone marrow cells is lower than simple irradiation group all the time, has significantly or highly significant significant difference (p＜0.05～0.01).

Each experimental mice neutron is listed in table 5 according to the variation of back splenocyte apoptosis rate.

The variation of different time splenocyte apoptosis rate after table 5. mouse neutron shines (x ± s)

Annotate: compare with control group ^*P＜0.05, ^*P＜0.01; Compare #P＜0.05, ##P＜0.01 with simple control group.

By table 5 as seen, neutron shone the back 1-28 days, and simple irradiation group and transgenosis group splenic lymphocyte apoptosis rate and control group significantly increase, and have highly significant significant difference (p＜0.01), shine the back the 7th day, and apoptosis rate all reaches maximum value.Compare with simple irradiation group, transgenosis group splenic lymphocyte apoptosis rate significantly reduces, and maintains a lower level all the time, has highly significant significant difference (p＜0.01).

Table 6 is listed in the variation of thymocyte apoptosis rate after each experimental mice neutron irradiation.

Table 6. mouse neutron is transferred the variation (x ± s) of the rate of dying according to back different time thymocyte

Annotate: compare with control group ^*P＜0.05, ^*P＜0.01; Compare #P＜0.05, ##P＜0.01 with simple control group.

By table 6 as seen, compare with aforementioned medullary cell and spleen lymphocyte, the thymic lymphocytes apoptosis rate significantly increases.Shining the back the 1st day, obviously increasing promptly appears in simple irradiation group thymic lymphocytes apoptosis rate, and reaches maximum value in the 7th day, falls after rise in the 14th day, still maintains a higher relatively level during to the 28th day.And the 1st day apoptosis rate of transgenosis group thymic lymphocytes is lower than control group slightly, begins to occur obvious rising in the time of the 7th day, then descends gradually, returns to lower level in the time of the 28th day.In whole experiment, transgenosis group thymic lymphocytes apoptosis rate is lower than simple irradiation group all the time, has significantly or highly significant significant difference (p＜0.05～0.01).

Annotate: statistical analysis is to use SAS 9.0 softwares to carry out other t check of two sample difference in means in the experiment.

Above embodiment has proved: in the acute severe radiotherapy damage that neutron or gamma-rays cause, the pprI genophore is entered in the mouse body by the live body electrotransfection as medicine, can effectively reduce mouse death rate, significantly reduce the quantity and the degree of leukopenia, significantly reduce medullary cell, spleen and thymic lymphocytes apoptosis rate, can obviously alleviate liver and kidney oedema and downright bad scope and degree, promote the reparation of hepatic and renal tissue, the severe radiotherapy damage is had significant anti-putting and therapeutic action.

SEQUENCE?LISTING

＜110〉University Of Suzhou

＜120〉a kind of eukaryon recombinant plasmid that contains prokaryon gene pprI and uses thereof

<160>2

<170>PatentIn?version?3.5

<210>1

<211>853

<212>DNA

＜213〉radioresistant bacterium (Deinococcus radiodurans)

<400>1

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ccaacgtcag?ccccccttgc?ccctctgggg?taaggggcgg?ggggatgggg?ccaaaagcta?180

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<210>2

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＜213〉radioresistant bacterium (Deinococcus radiodurans)

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cgagttgatg?aggatgacgt?tgtgctcggg?gtcgaacgcc?ccgtcgcgcc?agccgagcgg?840

catga 845

Claims (4)

1. intestinal bacteria, it is characterized in that: described intestinal bacteria are DH5 α/pCMV-HA-pprI, and its deposit number is: CCTCC NO:M208253.

2. recombinant plasmid, it is characterized in that: described recombinant vectors is pCMV-HA-pprI, the method for preparing described recombinant plasmid may further comprise the steps:

(1) being upstream primer with 5 '-ATGCCCAGTGCCAACGTCAGCCCCCCTT-3 ', is downstream primer with 5 '-TCACTGTGCAGCGTCCTGCGGCTCGTCC-3 ', is template with D.radiodurans R1 genome, and pcr amplification obtains the pprI gene;

(2) recycling step (1) gained PCR product pprI gene is connected with the pGEM-T carrier, will connect product transformed into escherichia coli DH5 α, selects positive colony then, extracting plasmid pGEM-T-pprI;

(3) be upstream primer with 5 '-TCGAATTCCCAGTGCCAACGTCAGCCCCCCTTGC-3 ', with 5 '-TTCTCGAGTTTCACTGTGCAGCGTCCTGCGGCTC-3 ' is downstream primer, with the pGEM-T-pprI plasmid that checks order correct is template, use pcr amplification, and the rubber tapping purifying reclaims the purpose fragment;

(4) with EcoRI enzyme and XhoI enzyme pprI fragment and pCMV-HA carrier are carried out double digestion, after purifying enzyme is cut product, with the T4 ligase enzyme pprI fragment is connected with the pCMV-HA carrier and obtains the pCMV-HA-pprI recombinant plasmid.

3. the application of the described recombinant plasmid of claim 2 in the preparation antiradiation injury medicine.

4. a medicine that prevents or treat radiation injury is characterized in that: contain the described recombinant plasmid of claim 2.

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